He Kuifu, Ma Yuechao, Du Shanshan, Xie Xixian, Xu Qingyang, Chen Ning
College of Biotechnology, Tianjin University of Science and Technology, Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin 300457, China.
Wei Sheng Wu Xue Bao. 2012 Jun 4;52(6):718-25.
To study the effects of overexpression of key enzyme genes (prs, purF and guaB) on guanosine production in Bacillus amyloliquefaciens TA208.
The prs, purF, guaB and prs-purF genes were inserted into constructed expression plasmid PBE43. All these constructed plasmids were electroporated into B. amyloliquefaciens TA208. The transcriptional level of various genes in the resulting strains was tested by real-time quantitative PCR. The activity of inosine 5'-monophosphate dehydrogenase in the resulting strains was detected. Finally, cell growth, glucose consumption and guanosine production of 4 engineering strains along with control strain were examined.
The transcriptional analysis showed that overexpression of prs, purF and guaB gene accompanied by their own transcription level up-regulated. Overexpression of prs or purF genes alone slightly down-regulated the transcriptional level of purine operon, but overexpression of guaB gene independently did not disturb the transcription of prs gene and purine operon. Enzyme activity analysis showed that overexpression of prs or purF gene did not change the activity of inosine 5'-monophosphate dehydrogenase and its activity increased by 126% through overexpression of guaB gene. Finally, by fermentation flask test, we found that overexpression of prs and purF gene alone could not promote guanosine accumulation. However, overexpression of guaB gene resulted in an increase in the production of guanosine, which was 20.7% higher than the control strain. The guanosine concentration and the conversion ratio from glucose to guanosine in the host strain containing co-expression plasmid were 14.4% and 6.8% higher than the control strain.
Overexpression of guaB gene could enhance the guanosine yield in the culture broth. However, for prs and purF gene, only co-expression of them could lead to a significant improvement of guanosine production in B. amyloliquefaciens. It should provide a valuable insight into the construction of industrially important strains for guanosine production by metabolic engineering.
研究关键酶基因（prs、purF和guaB）过表达对解淀粉芽孢杆菌TA208中鸟苷产量的影响。
将prs、purF、guaB和prs-purF基因插入构建好的表达质粒PBE43中。将所有构建好的质粒电穿孔导入解淀粉芽孢杆菌TA208。通过实时定量PCR检测所得菌株中各种基因的转录水平。检测所得菌株中5'-肌苷酸脱氢酶的活性。最后，检测4株工程菌株和对照菌株的细胞生长、葡萄糖消耗及鸟苷产量。
转录分析表明，prs、purF和guaB基因过表达伴随其自身转录水平上调。单独过表达prs或purF基因会轻微下调嘌呤操纵子的转录水平，但单独过表达guaB基因不会干扰prs基因和嘌呤操纵子的转录。酶活性分析表明，过表达prs或purF基因不会改变5'-肌苷酸脱氢酶的活性，而过表达guaB基因可使其活性提高126%。最后，通过摇瓶试验发现，单独过表达prs和purF基因不能促进鸟苷积累。然而，过表达guaB基因导致鸟苷产量增加，比对照菌株高20.7%。含有共表达质粒的宿主菌株中鸟苷浓度和葡萄糖到鸟苷的转化率比对照菌株分别高14.4%和6.8%。
过表达guaB基因可提高培养液中鸟苷产量。然而，对于prs和purF基因，只有它们共表达才能显著提高解淀粉芽孢杆菌中鸟苷的产量。这应为通过代谢工程构建用于鸟苷生产的重要工业菌株提供有价值的见解。
